Biomaterial derived from vertebrate liver tissue

ABSTRACT

A tissue graft composition comprising liver basement membrane is described. The graft composition can be implanted to replace or induce the repair of damaged or diseased tissues.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 09/319,781, filed on Jun. 10, 1999, now U.S. Pat.No. 6,379,710, which is a U.S. national phase counterpart ofinternational application serial No. PCT/US97/22727, filed Dec. 10,1997, which claims priority to U.S. Provisional Application serial No.60/032,680, filed on Dec. 10, 1996.

This invention was made with U.S. Government support under Grant#HD-31425 awarded by the National Institute of Health. The U.S.Government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to a tissue graft composition and methodsfor its preparation and use. More particularly, the present invention isdirected to non-immunogenic tissue graft compositions comprising thebasement membrane of liver and the use of same to promote endogenoustissue growth in vivo and to support the growth and differentiation ofeukaryotic cells cultured in vitro.

BACKGROUND AND SUMMARY OF THE INVENTION

There has been much research effort directed to finding natural andsynthetic materials having the requisite properties for use as tissuegrafts. Surprisingly, it has been found that basement membranes (stroma)prepared from liver tissue of warm-blooded vertebrates by removingcellular components of the liver tissue exhibit certain mechanical andbiotropic properties similar to that which has been reported forintestinal submucosal tissue in U.S. Pat. Nos. 4,902,508; 5,281,422; and5,275,826. It can be substituted for intestinal submucosa tissue inmost, if not all, of the applications previously reported for intestinalsubmucosa, including enhancing wound healing, promoting endogenoustissue growth, stimulating cell proliferation and inducing celldifferentiation.

The basement membrane of the liver is an extracellular matrix distinctfrom submucosal extracellular matrices. The liver basement membrane doesnot support an overlaying mucosa and is devoid of the laminate tissuestructure in which submucosal extracellular matrices reside. The liverplays a central role in numerous regulatory processes in the body,including glucose metabolism, insulin regulation, anabolic processes forthe musco-skeletal system and central nervous system, and themaintenance of appropriate levels of circulating proteins essential forday to day homeostasis.

In one embodiment of the present invention, liver basement membranes areused to manufacture a non-immunogenic tissue graft composition for usein the repair of damaged or diseased tissues. The tissue graftcomposition of the present invention comprises the basement membrane oforgan tissue of a warm-blooded vertebrate, for example, liver tissue,substantially free, preferably devoid, of all cells (e.g., hepatocytesand bile ductal cells) of said warm-blooded vertebrate. The presenttissue graft composition can be implanted, or fluidized and injected,into a vertebrate host to contact damaged or defective tissues andinduce the repair or replacement of said tissues. The compositions ofthe present invention can also be applied as a component of a wounddressing (ointment or bandage) in fluidized or solid form for topicalapplication to promote wound healing. Alternatively, the liver tissuederived extracellular matrix can be utilized as a cell growth substratefor growing eukaryotic cells in vitro.

DETAILED DESCRIPTION OF THE INVENTION

The tissue graft composition of the present invention comprises liverbasement membrane prepared by separating same from the nativelyassociated cellular components of liver tissue of a warm-bloodedvertebrate. The preparative techniques described below provide anextracellular matrix composition consisting essentially of liverbasement membrane substantially free of any cellular components. Thesecompositions are referred to herein generically as liver basementmembrane(s) (LBM). Other organ tissue sources of basement membrane foruse in accordance with this invention include spleen, lymph nodes,salivary glands, prostate, pancreas and other secreting glands.

Basement membrane for use in the graft composition of this invention istypically prepared from liver tissue harvested from animals raised formeat production, including, for example, pigs, cattle and sheep or otherwarm-blooded vertebrates. Thus, there is an inexpensive commercialsource of liver tissue for use in preparation of the tissue graftcompositions in accordance with the present invention. In accordancewith one embodiment, a composition comprising liver basement membranesis prepared from liver tissue of a warm-blooded vertebrate. Thiscomposition is useful as a non-immunogenic tissue graft capable ofinducing endogenous tissue growth when implanted in warm-bloodedvertebrates. In one embodiment, the composition comprises anextracellular matrix consisting essentially of liver basement membranedevoid of endogenous cells associated with the source vertebrate livertissue used to prepared the composition.

The preparation of liver basement membrane from of liver tissue of awarm-blooded vertebrate in accordance with the present invention iscarried out by removing the cellular components from liver tissue.Ideally the process is carried out to separate the cells from thebasement membranes without damaging, or at least minimizing disruptionor damage to, the basement membrane tissue. Removal of the cellularcomponents from the liver extracellular matrix allows the preparation ofa graft composition that is non-immunogenic, and thus does not induce ahost immune response when the graft composition is implanted into ahost. In general, the method for preparing a tissue graft compositionfrom warm-blooded vertebrate liver tissue comprising the steps oftreating the liver tissue with a cell dissociation solution for a periodof time sufficient to release the cellular components of the livertissue from the extracellular components without substantial disruptionof the extracellular components, and separating the cellular componentsfrom said extracellular components. Typically the cell dissociationsolution comprises a chaotropic agent or an enzyme or both.

The first step in preparing LBM in accordance to one embodiment of thepresent invention comprises slicing a segment of liver tissue intopieces (e.g., strips or sheets) to increase the surface area-to-volumeratio of the liver tissue. In one embodiment the liver tissue is slicedinto a series of sheets each having a thickness of about 50 to about 500microns, more preferably about 250 to about 300 microns. Freshlyharvested liver tissue can be sliced using a standard meat slicer, orthe tissue can be frozen and sliced with a cryomicrotone. The thinpieces of liver tissue are then treated with a solution that releasescomponent liver cells from the associated extracellular basementmembrane matrix.

In accordance with one embodiment the liver tissue is treated with asolution comprising an enzyme, for example, a protease, such as trypsinor pepsin. Because of the collagenous structure of the liver basementmembrane and the desire to minimize degradation of the membranestructure during cell dissociation, collagen specific enzyme activityshould be minimized in the enzyme solutions used in thecell-dissociation step. In addition, the liver tissue is typically alsotreated with a calcium chelating agent or chaotropic agent such as amild detergent such as Triton 100. Thus, in one embodiment of thisinvention liver tissue is treated by suspending slices or strips of thetissue in a cell-dissociation solution containing enzyme(s) andchaotropic agent(s). However, the cell dissociation step can also beconducted using a calcium chelating agent or chaotropic agent in theabsence of an enzymatic treatment of the tissue.

In one preferred embodiment the cell-dissociation step is carried out bysuspending liver tissue slices in an agitated solution containing about0.05 to about 2%, more typically about 0.1 to about 1% by weightprotease, optionally containing a chaotropic agent or a calciumchelating agent in an amount effective to optimize release andseparation of cells from the basement membrane without substantialdegradation of the membrane matrix.

After contacting the liver tissue with the cell-dissociation solutionfor a time sufficient to release all cells from the matrix, theresulting liver basement membrane is rinsed one or more times withsaline and optionally stored in a frozen hydrated state or a partiallydehydrated state until used as described below. The cell-dissociationstep may require several treatments with the cell-dissociation solutionto release substantially all cells from the basement membrane. In oneembodiment liver tissue is treated with a protease solution to removethe component cells, and the resulting extracellular matrix material(basement membrane) is further treated to remove or inhibit any residualenzyme activity. For example, the resulting basement membrane can beheated or treated with one or more protease inhibitors.

Liver basement membrane in accordance with this invention can befluidized (converted to an injectable or powder form) in a mannersimilar to the preparation of fluidized intestinal submucosa, asdescribed in U.S. Pat. No. 5,275,826 the disclosure of which isexpressly incorporated herein by reference. Liver basement membrane(devoid of cells from the source liver tissue) is comminuted by tearing,cutting, grinding, shearing and the like. Grinding the liver basementmembrane in a frozen or freeze-dried state is preferred although goodresults can be obtained as well by subjecting a suspension of liverbasement membrane to treatment in a high speed (high shear) blender anddewatering, if necessary, by centrifuging and decanting excess water.Additionally, the comminuted fluidized tissue can be solubilized byenzymatic digestion with a protease, for example a collagenase and orother appropriate enzyme, such as glycanase, or other enzyme thatdisrupts the matrix structural components, for a period of timesufficient to solubilize said tissue and form a substantiallyhomogeneous solution.

The present invention also contemplates the use of powder forms of liverbasement membrane. In one embodiment a powder form of liver basementmembrane is prepared by pulverizing liver basement membrane submucosatissue under liquid nitrogen to produce particles ranging in size from0.1 to 1 mm². The particulate composition is then lyophilized overnightand sterilized to form a solid substantially anhydrous particulatecomposite. Alternatively, a powder form of liver basement membrane canbe formed from fluidized liver basement membranes by drying thesuspensions or solutions of comminuted/liver basement membrane. Thedehydrated forms have been rehydrated and used as cell culturesubstrates as described below without any apparent loss of their abilityto support cell growth.

To determine the components of the isolated liver basement membranes ofthe present invention, the membranes have been extracted and theisolated fractions analyzed by Western blot analysis. LBM was extractedwith guanidine hydrochloride or urea, as described in Example 4 andWestern blot analysis, using antibodies directed against variousspecific growth factors, indicated the presence of basic growthfibroblast growth factor (bFGF), hepatocyte growth factor (HGF) andepidermal growth factor

The present liver basement membrane compositions may be sterilized usingconventional sterilization techniques including tanning withglutaraldehyde, formaldehyde tanning at acidic pH, ethylene oxidetreatment, propylene oxide treatment, gas plasma sterilization, gammaradiation, and peracetic acid sterilization. A sterilization techniquewhich does not significantly weaken the mechanical strength andbiotropic properties of the material is preferably used. For instance,it is believed that strong gamma radiation may cause loss of strength inthe graft material. Preferred sterilization techniques include exposingthe graft to peracetic acid, low dose gamma irradiation and gas plasmasterilization; peracetic acid sterilization being the most preferredmethod. In particular, LBM has been disinfected and sterilized throughthe use of either peracetic acid or one megarad of gamma irradiationwithout adversely effecting the mechanical properties or biologicalproperties of the tissue. The treatment with peracetic acid is conductedat a pH of about 2 to about 5 in an aqueous ethanolic solution (2-10%ethanol by volume) at a peracid concentration of about 0.03 to about0.5% by volume. Typically, after the graft composition has beensterilized, the composition is wrapped in a porous plastic wrap andsterilized again using electron beam or gamma irradiation sterilizationtechniques.

In accordance with one embodiment, liver basement membrane is used as,or used to prepare, tissue graft compositions of the present invention.Such tissue graft compositions lend themselves to a wide variety ofsurgical applications relating to the repair or replacement of damagedtissues, including, for example the repair of connective tissues.Connective tissues for the purposes of the present invention includesbone, cartilage, muscle, tendons, ligaments, and fibrous tissueincluding the dermal layer of skin.

In accordance with this invention, the present tissue graft compositionsare used advantageously to induce the formation of endogenous tissue ata desired site in a warm blooded vertebrate. Compositions comprising anextracellular matrix, consisting essentially of liver basement membrane,can be administered to a vertebrate host in an amount effective toinduce endogenous tissue growth at a site in the host in need of samedue to the presence of damaged or diseased tissue. The present livertissue derived tissue graft compositions can be administered to the hostin either solid form, by surgical implantation, or in fluidized form, byinjection.

The liver basement membrane segments can be used in accordance with thisinvention as a tissue graft construct for use in the repair orreplacement of connective tissues using the same procedures describedfor use of intestinal submucosa in U.S. Pat. Nos. 5,281,422 and5,352,463, each expressly incorporated herein by reference.

The tissue graft compositions formed and used in accordance with thisinvention, upon implantation, undergo biological remodeling. They serveas a rapidly vascularized matrix for supporting the growth of newendogenous connective tissue. When used as a tissue graft material liverbasement membrane is expected to be trophic for host tissues with whichit is attached or otherwise associated in its implanted environment.

The liver basement membrane graft composition can be formed in a varietyof shapes and configurations, for example, to serve as a ligament ortendon replacement or a patch for a broken or severed tendon orligament. Preferably, the segment is shaped and formed to have a layeredor even a multilayered configuration with at least the opposite endportions and/or opposite lateral portions being formed to have multiplelayers of the graft material to provide reinforcement for attachment tophysiological structures, including bone, tendon, ligament, cartilageand muscle. In a ligament replacement application, opposite ends areattached using standard surgical technique to first and second bones,respectively, the bones typically being articulated as in the case of aknee joint.

The end portions of the liver basement membrane graft composition can beformed, manipulated or shaped to be attached, for example, to a bonestructure in a manner that will reduce the possibility of graft tearingat the point of attachment. Preferably the material can be folded or toprovide multiple layers for gripping, for example, with spiked washersor staples.

Alternatively, the liver basement membrane graft material may be foldedback on itself to join the end portions to provide a first connectiveportion to be attached, for example, to a first bone and a bend in theintermediate portion to provide a second connective portion to beattached to a second bone articulated with respect to the first bone.For example, one of the end portions may be adapted to be pulled througha tunnel in, for example, the femur and attached thereto, while theother of the end portions may be adapted to be pulled through a tunnelin the tibia and attached thereto to provide a substitute for thenatural cruciate ligament, the segment being adapted to be placed undertension between the tunnels to provide a ligament function, ie, atensioning and positioning function provided by a normal ligament.

The liver basement membranes of the present invention have beenimplanted in rabbits and in dogs to serve as Achilles tendon replacementgraft constructs. Two rabbits and two dogs were each implanted withAchilles tendon replacement LBM graft constructs using a similarprocedure as used for intestinal submucosal tissue as describe in U.S.Pat. No. 4,902,508. The experiments demonstrated that LBM graftconstructs could support the regeneration of the Achilles tendon.

During preparation of the liver basement membrane, the tissue is cut orsliced into pieces/slices. After the cell-dissociation processing stepthe individual segments of liver basement membrane can be overlappedwith one another and bonded together using standard techniques known tothose skilled in the art, including the use of sutures, crosslinkingagents, and adhesives or pastes. Alternatively, in one embodiment, theoverlapped layers of submucosal tissue are fused to one another byapplying pressure to the overlapped regions under dehydratingconditions. The term “dehydrating conditions” is defined to include anymechanical or environmental condition which promotes or induces theremoval of water from the submucosal tissue. To promote dehydration ofthe compressed submucosal tissue, at least one of the two surfacescompressing the tissue is water permeable. Dehydration of the tissue canoptionally be further enhanced by applying blotting material, heatingthe tissue or blowing air across the exterior of the compressingsurfaces. Accordingly, multilayer liver basement membrane constructs canbe prepared to provide tissue graft compositions of enhanced strength.

In addition, by overlapping a portion of one piece of liver basementmembrane with a portion of at least one additional piece of liverbasement membrane and bonding the overlapped layers to one another,large area sheets of liver basement membrane can be formed. In oneembodiment, during formation of the large area sheets of tissue,pressure is applied to the overlapped portions under dehydratingconditions by compressing the overlapped tissue segments between twosurfaces. The two surfaces can be formed from a variety of materials andin any shape depending on the desired form and specification of thetargeted graft construct. Typically the two surfaces are formed as flatplates but they can also include other shapes such as screens, opposedcylinders or rollers and complementary nonplanar surfaces. Each of thesesurfaces can optionally be heated or perforated. In preferredembodiments at least one of the two surfaces is water permeable. Theterm water permeable surface as used herein includes surfaces that arewater absorbent, microporous or macroporous. Macroporous materialsinclude perforated plates or meshes made of plastic, metal, ceramics orwood.

The liver basement membrane can be compressed in accordance with oneembodiment by placing the overlapped portions of the strips ofcell-dissociated liver membrane on a first surface and placing a secondsurface on top of the exposed membrane surface. A force is then appliedto bias the two surfaces towards one another, compressing the membranecomposition between the two surfaces. The biasing force can be generatedby any number of methods known to those skilled in the art including thepassage of the apparatus through a pair of pinch rollers (the distancebetween the surface of the two rollers being less than the originaldistance between the two plates), the application of a weight on the topplate, and the use of a hydraulic press or the application ofatmospheric pressure on the two surfaces.

In one preferred embodiment, a multi-layered liver basement membranegraft material is prepared without the use of adhesives or chemicalpretreatments by compressing at least the overlapped portions ofsubmucosal tissue under conditions that allow dehydration of thematerial concurrent with the compression of the tissue. To promotedehydration of the compressed material, at least one of the two surfacescompressing the tissue is water permeable. Dehydration can optionally befurther enhanced by applying blotting material, heating the material orblowing air across the exterior of the two compressing surfaces. Thecompressed multi-layered liver basement membrane material can be removedfrom the two surfaces as a unitary compliant large area graft construct.The construct can be further manipulated (i.e., cut, folded, sutured,etc.) to suit various medical applications where the liver basementmembrane material is required.

A vacuum can optionally be applied to liver basement membrane during thecompression procedure. The applied vacuum enhances the dehydration ofthe tissue and may assist the compression of the tissue. Alternativelythe application of a vacuum may provide the sole compressing force forcompressing the overlapped portions of the multiple layers of liverbasement membranes. For example, in one embodiment the overlapped liverbasement membrane is laid out between two surfaces, preferably one ofwhich is water permeable. The apparatus is covered with blottingmaterial, to soak up water, and a breather blanket to allow air flow.The apparatus is then placed in a vacuum chamber and a vacuum isapplied, generally ranging from 35.6-177.8 cm of Hg (0.49-2.46 Kg/cm²)and more preferably the vacuum applied is approximately 129.5 cm of Hg(1.76 Kg/cm²). Optionally a heating blanket can be placed on top of thechamber to heat the liver basement membrane during compression. Chamberssuitable for use in this embodiment are known to those skilled in theart and include any device that is equipped with a vacuum port. Theresulting drop in atmospheric pressure coacts with the two surfaces tocompress the tissue and simultaneously dehydrate the compressed tissue.

In an alternative embodiment of the present invention, liver basementmembrane can be utilized in a method and composition for supporting theproliferation and induction of tissue differentiation of eukaryoticcells cultured in vitro. Generally the method comprises the step ofcontacting eukaryotic cells, in vitro, with a liver basement membranecomposition under conditions conducive to eukaryotic cell growth. Theterm “contacting” as used herein with reference to cell culture isintended to include both direct and indirect contact, for example influid communication, of the liver basement membrane composition and thecultured cells. The term “conditions conducive to eukaryotic cellgrowth” as used herein refers to the environmental conditions, such assterile technique, temperature and nutrient supply, that are consideredoptimal for eukaryotic cell growth under currently available cellculture procedures. Although optimum cell culture conditions used forculturing eukaryotic cells depend somewhat on the particular cell type,cell growth conditions are generally well known in the art. However anumber of differentiated cell types are still considered difficult toculture (i.e., islets of Langerhans, hepatocytes, chondrocytes,osteoblasts, etc.).

Applicants have discovered that compositions comprising liver basementmembrane prepared in accordance with this invention can be used forsupporting growth or proliferation of eukaryotic cells in vitro. Inaccordance with one embodiment a liver tissue derived composition forsupporting the growth of a cell population is prepared from liver tissueof a warm-blooded vertebrate. The composition comprises isolated liverbasement membrane devoid of source liver tissue endogenous cells andadded nutrients to support the growth of said cell population in vitro.In addition fluidized forms of liver basement membrane can be used tocoat culture-ware with a matrix comprising liver basement membranedevoid of source liver tissue endogenous cells. Thus liver basementmembrane can be used as a cell growth substrate in a variety of forms,including a sheet-like configuration, as a gel matrix, as an additivefor art-recognized cell/tissue culture media, or as coating forculture-ware to provide a more physiologically relevant substrate thatsupports and enhances the proliferation of cells.

The liver basement membrane, due to its honeycomb-like structure (thatwhich remains after cell-dissociation) provides a high surface area forcell adhesion and also induces cell differentiation. Scanning electronimages indicate that the isolated liver basement membrane is veryporous. When fetal rat cells are cultured on liver basement membranesthat are retained in their nature three dimensional shape, scanningelectron images reveal that the fetal rat cells form confluent sheets onthe liver cell substrate and also invade into the LBM matrix. Themembrane material is preferably sterilized prior to use in cell cultureapplications, however nonsterile material can be used if antibiotics areincluded in the cell culture system.

In one preferred embodiment cells are seeded directly onto sheets ofliver basement membrane under conditions conducive to eukaryotic cellproliferation. The highly porous nature of the liver basement membraneallow diffusion of cell nutrients throughout the membrane matrix. Thus,cells can be cultured on or within the liver basement membrane matrix.

In another embodiment of the present invention, cell growth substratesare formed from fluidized forms of liver basement membrane. Thefluidized tissue can be gelled to form a solid or semi-solid matrix. Theviscosity of fluidized tissue for use in accordance with this inventioncan be manipulated by controlling the concentration of the tissuecomponent and the degree of hydration. The viscosity can be adjusted toa range of about 2 to about 300,000 cps at 25° C. Higher viscosityformulations, for example, gels, can be prepared from the digestsolutions by adjusting the pH of such solutions to about 6.0 to about7.4. Eukaryotic or prokaryotic cells can then be seeded directly on thesurface of the matrix and cultured under conditions conducive toeukaryotic cell proliferation.

The cell growth substrates of the present invention can be combined withnutrients; including minerals, amino acids, sugars, peptides, proteins,or glycoproteins that facilitate cellular proliferation, such as lamininand fibronectin and growth factors such as epidermal growth factor,platelet-derived growth factor, transforming growth factor beta, orfibroblast growth factor. In one embodiment fluidized or powder forms ofliver basement membrane can be used to supplement standard eukaryoticculture media to enhance the standard media's capacity for sustainingand inducing the proliferation of cells cultured in vitro.

In accordance with the present invention there is provided a cellculture composition for supporting growth in vitro of an eukaryotic cellpopulation in combination with liver basement membrane of a warm-bloodedvertebrate. The composition comprises liver basement membranesubstantially free of the original associated endogenous cells. Thecomposition can further comprise nutrients, and growth factors requiredfor optimal growth of the cultured cells. The liver basement membranecell culture substrate can be used with commercially available cellculture liquid media (both serum based and serum free). Proliferatingcells can either be in direct contact with the liver basement membraneor they can simply be in fluid communication with the liver basementmembrane.

It is anticipated that cell growth compositions utilizing the liverbasement membrane composition of the present invention can be used tostimulate proliferation of undifferentiated stems cells as well asdifferentiated cells such as islets of Langerhans, hepatocytes andchondrocytes. Furthermore, the described cell growth composition isbelieved to support the growth of differentiated cells while maintainingthe differentiated state of such cells. Several primary cell lines havebeen grown on LBM derived cell culture substrates in vitro, includingprimary cell lines derived from the cruciate ligament. Primary celllines derived from the cruciate ligament show an approximate doubling ofthe growth rate compared to when these cells are grown on plastic.

It is anticipated that liver basement membrane is capable of inducinghost tissue proliferation, remodeling and regeneration of appropriatetissue structures upon implantation in a number of microenvironments invivo (e.g., tendon, ligament; bone, articular cartilage, artery, andvein). In one embodiment of the present invention the tissue replacementcapabilities of graft compositions comprising liver basement membrane ofwarm-blooded vertebrates are further enhanced or expanded by seeding thetissue with various cell types, prior to implantation. For example, aliver basement membrane derived cell culture substrate may be seededwith endothelial cells or keratinocytes for use as a vascular graft orskin replacement, respectively. Alternatively, the liver basementmembrane can be seeded with mesenchymal cells (stem cells) initially forexpansion of the cell population and thereafter for implantation into ahost. Liver basement membrane can also serve as a delivery vehicle,either in fluidize form or in its native solid form, for introducingvarious cell populations, including genetically modified cells, to aspecific location in a host. Optionally, after the liver basementmembrane have been seeded with eukaryotic cells, the graft compositioncan be subjected to conditions conducive to the proliferation ofeukaryotic cells to further expand the population of the seeded cellsprior to implantation of the graft into the host.

In another embodiment, compositions comprising liver basement membraneand a proliferating cell population can be encapsulated in abiocompatible matrix for implantation into a host. The encapsulatingmatrix can be configured to allow the diffusion of nutrients to theencapsulated cells while allowing the products of the encapsulated cellsto diffuse from the encapsulated cells to the host cells. Suitablebiocompatible polymers for encapsulating living cells are known to thoseskilled in the art. For example a polylysine/alginate encapsulationprocess has been previously described by F. Lim and A. Sun (Science Vol.210 pp. 908-910). Indeed, the present liver basement membranecomposition itself could be used advantageously to encapsulate aproliferating cell population in accordance with this invention forimplantation as an artificial organ.

EXAMPLE 1

Preparation of Liver Basement Membrane

2 mM EDTA Chaotropic Solution Used In The Experiment 140 mM NaCl 5 mMKCl 0.8 mM MgSO₄ 0.4 mM KH₂HPO₄ 2 mM EDTA 25 mM NaHCO₃

Procedure

Preparation of Liver Slices

Liver frozen in −70° C. was sliced with a cryomicrotone to a thicknessof about 50 μM. The slices of liver tissue were then subjected toenzymatic treatment (trypsin) with a chaotropic solution (samples 1 and2), with enzyme alone (samples 3 and 4), or with a chaotropic solutionalone (sample 5), as indicated below.

Sample # Treatment 1) 0.05% Trypsin in 2 mM EDTA solution 2) 0.1%Trypsin in 2 mM EDTA solution 3) 0.05% Trypsin in 2 mM PBS 4) 0.1%Trypsin in 2 mM PBS 5) 2 mM EDTA solution

Liver slices were placed in five 50 ml tubes, each of which contained 25mL of a different buffered enzyme treatment solution. The liver tissuewas incubated at 37° C. in water bath with gentle shaking for 1 hour.The liver slices were washed twice with PBS with agitation/shaking for 1hour at room temperature. The above enzymatic treatment steps wererepeated three times.

The wash buffers were collected and spin them down in 2000 rpm for 10min. The pellet was suspended and an equal amount of trypan blue wasadded to identify any remaining cells. The material was checked forpresence of cells under microscope.

EXAMPLE 2

Mechanical Properties of Isolated Liver Basement Membrane

Porosity of a graft material is typically measured in terms of ml ofwater passed per cm²min⁻¹ at a pressure of 120 mm Hg. The average“porosity index” established for two separate specimens of LBM was 1162.The suture retention strength of LBM is approximately 68 grams. Thematerial appears to be anisotropic, with the suture strength beingapproximately the same in all directions.

EXAMPLE 3

Growth and Differentiation of Various Cell Lines on Liver BasementMembrane

In the present study, the growth and differentiation of three differentcell lines on liver basement membrane (LBM) was investigated. Thesetested cell lines included Swiss 3T3, a fibroblast cell line, ROS.17/2.8, an osteosarcoma cell line and PC12, a neuronal cell line. Allcells were seeded on a collagen coated plate. The seeding densities forSwiss 3T3 and ROS cells was 20×10⁴ cells/ml, whereas the PC12 was seededat 5×10⁴ cells/ml. In order to compare the growth and differentiation ofthese cells lines on LBM and small intestinal submucosa (SIS), the samecells with same densities were also seeded on SIS. In addition, apositive control for PC12 cells was achieved by adding nerve growthfactor (NGF) 50 ng/ml to the cells. All experiments were carried out induplicate. After five days one sample from each cell line seeded on SISand LBM was prepared for histology.

Preliminary observations indicate growth of all three cell lines anddifferentiation of ROS and PC12 cell lines on both substrates. A lightmicroscopic observation of PC12 cells indicated a greater degree ofdifferentiation on LBM compared to the cells grown on SIS. In addition,ROS and 3T3 cells appeared to grow/differentiate as well as they do onSIS. Due to the transparent nature of LBM, it was difficult to quantifythe growth and differentiation of the cells, but the histologicalexamination will allow a more detailed assessment.

EXAMPLE 4

Preparation of Extracts of LBM

Extraction buffers used for these studies included 4 M guanidine and 2 Murea each prepared in 50 mM Tris-HCl, pH 7.4. The powder form of LBM wassuspended in the relevant extraction buffer (25% w/v) containingphenylmethyl sulphonyl fluoride, N-ethylmaleimide, and benzamidine(protease inhibitors) each at 1 mM and vigorously stirred for 24 hoursat 4° C. The extraction mixture was then centrifuged at 12,000×g for 30minutes at 4° C. and the supernatant collected. The insoluble materialwas washed briefly in the extraction buffer, centrifuged, and the washcombined with the original supernatant. The supernatant was dialyzedextensively in Spectrapor tubing (MWCO 3500, Spectrum MedicalIndustries, Los Angeles, Calif.) against 30 volumes of deionized water(9 changes over 72 hours). The dialysate was centrifuged at 12,000×g toremove any insoluble material and the supernatant was used immediatelyor lyophilized for long term storage.

Western blot analysis with antibodies specific for bFGF HGF and EGFdetected a corresponding reactive band for each of the antibodies,confirming the presence of these growth factors in LBM.

What is claimed is:
 1. A method for inducing the formation of endogenoustissue at a site in need of endogenous tissue growth in a warm-bloodedvertebrate, said method comprising implanting a graft compositioncomprising an extracellular matrix consisting essentially of basementmembrane of liver tissue of a warm-blooded vertebrate in an amounteffective to induce endogenous tissue growth at the site the graftcomposition is administered, said liver basement membrane being devoidof endogenous cells associated with said liver tissue.
 2. The method ofclaim 1 wherein the basement membrane is fluidized and the graftcomposition is administered by injection into the warm-bloodedvertebrate.
 3. The method of claim 1 wherein the graft composition isadministered by surgically implanting the composition into thewarm-blooded vertebrate.
 4. The method of claim 1 wherein the endogenouscells are dissociated with a cell dissociation solution.
 5. The methodof claim 4 wherein the cell dissociation solution comprises a chaotropicagent.
 6. The method of claim 4 wherein the cell dissociation solutioncomprises a protease.
 7. The method of claim 4 wherein the celldissociation solution comprises EDTA and trypsin.
 8. The method of claim4 wherein the liver tissue is sliced into sheets or strips of livertissue before the liver tissue is treated with the dissociationsolution.
 9. The method of claim 8 wherein the liver tissue is slicedinto sheets or strips having a thickness of up to about 500 μ.
 10. Themethod of claim 1 further comprising the step of comminuting theextracellular matrix material to form a fluidized graft composition. 11.The method of claim 10 further comprising the step of enzymaticallydigesting the extracellular matrix material to form a fluidized graftcomposition.
 12. The method of claim 1 further comprising the step ofenzymatically digesting the extracellular matrix material to form afluidized graft composition.
 13. The method of claim 10 furthercomprising the step of drying the fluidized graft composition to theform of a dry powder.
 14. The method of claim 11 further comprising thestep of drying the fluidized graft composition to the form of a drypowder.